Summary

RMgm-685
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0932000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Details mutation: The mutant contains a mutated ron4 gene with two FRT sites
Details conditional mutagenesis: The FRTed sequence of ron4 is removed in the sporozoite stage by the Flp/FRT system
Transgene
Transgene not Plasmodium: FlpL recombinase of yeast (FlpL)
Promoter: Gene model: PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2; SSP-2)
3'UTR: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Insertion locus: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Insertion locus: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts; pbdhfr/ts)
Phenotype Sporozoite; Liver stage;
Last modified: 29 December 2011, 11:00
  *RMgm-685
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22177563
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone P. berghei NK65 TRAP/FlpL(-)F
Other information parent lineTRAP/FlpL(-)F is a P. berghei NK65 mutant (RMgm-683) that expresses the yeast FlpL recombinase under the control of the trap promoter. In addition it expresses GFP under the control of the constitutive hsp70 promoter. The mutant contains the dhfr/ts gene of P. berghei as a drug-selectable marker (PubMed: PMID: 21886105).
The mutant parasite was generated by
Name PI/ResearcherD. Giovannini; R. Menard
Name Group/DepartmentUnité de Biologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-685
Principal nameRON4/Cond
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteEvidence is presented that in the absence of RON4 expression ('RON4 silenced' sporozoites), sporozoites were not infective to hepatocytes in vitro.
Liver stageEvidence is presented that in the absence of RON4 expression ('RON4 silenced' sporozoites), sporozoites were not infective to hepatocytes in vitro.
Additional remarks phenotype

Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of RON4. The mutant expresses the yeast FlpL recombinase under the control of the trap promoter and contains a mutated ron4 gene that contains two FRT sites. The 3' UTR of ron4 is replaced with a 3' UTR of trap which contains the two FRT sites. This mutant has been generated by replacement of the 3'UTR of the endogenous ron4 gene by an 'FRTed' ron4 gene in mutant RMgm-683 that expresses FlpL.

The ron4 locus locus is disrupted by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-683). Removal of the FRTed ron4 sequence has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the 3' UTR of ron4 sequence which was flanked by FRT sequences.

Protein (function)
Rhoptry protein 4 (RON4) is unique to and conserved in apicomplexans. RON4 forms a complex with different other proteins (e.g. AMA1, RON2, RON5) during invasion of the merozoite at the tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole. Unsuccessful attempts to disrupt the ron4 gene in P. berghei (RMgm-686) indicate an essential role during asexual blood stage proliferation.

Phenotype
Unsuccessful attempts to disrupt the ron4 gene in P. berghei (RMgm-686) indicate an essential role during asexual blood stage proliferation.

In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence RON4 expression specifically in sporozoites. Removal of the FRTed 3' UTR sequence of ron4 has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the ron4 sequence which was flanked by FRT sequences.

The phenotype analyses indicate that RON4 is  essential for sporozoite infectivity to liver cells. Evidence is presented that 'RON4-silenced' sporozoites were unable to invade hepetocytes in vitro.

Additional information
In the same study a conditional knock-out mutant' of AMA1 has been generated using the same approach as described here (see RMgm-684).  It is shown that in contrast to RON4, AMA1 is not essential for sporozoite invasion of hepatocytes and development of liver stages.

Other mutants
RMgm-686: Unsuccessful attempts to disrupt the ron4 gene


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0932000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Short description of the mutationThe mutant contains a mutated ron4 gene with two FRT sites
Inducable system usedFlp/FRT
Short description of the conditional mutagenesisThe FRTed sequence of ron4 is removed in the sporozoite stage by the Flp/FRT system
Additional remarks inducable system
Click to view information
Click to hide information
A conditional mutagenesis approach was utilized that uses stage-specific expression of a temperature-sensitive variant of the yeast recombinase, flippase (FlpL) to catalyze the excision of DNA placed between two Flp Recognition Target sequences (FRT) (see RMgm-683). FlpL catalyzes site-specific DNA recombination at a temperature range of 25°-30°C.
The ron4 locus was modified by replacing the 3' UTR of ron4 with a 3' UTR of trap which contains the two FRT sites. This mutant has been generated by replacement of the 3'UTR of the endogenous ron4 gene by an 'FRTed' ron4 gene in mutant RMgm-683 that expresses FlpL.
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameFlpL recombinase of yeast (FlpL)
Details of the genetic modification
Inducable system usedFlp/FRT
Additional remarks inducable system The mutant expresses the yeast FlpL recombinase under the control of the promoter of trap. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences.
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences.
The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences.
In the resulting mutant (TRAP/Flp(-), by integrating at the DHFR-TS locus, a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2; SSP-2
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite PBANKA_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences.
The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences.
In the resulting mutant (TRAP/Flp(-), by integrating at the DHFR-TS locus, a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene.

To make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant pbdhfr-ts gene
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts; pbdhfr/ts
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4